The Use Of Suspension Culture And Efficient Induction To Study The Somatic Embryo System Of Taxus Cuspidata Sieb. Et Zucc

In this study, the young shoots and seeds of Taxus cuspidata Sieb.et Zucc were sampledfor somatic embryo induction, proliferation, maturation and then factors including planthormone, illumination, andglutamine were tested for their effects on the somatic embryoinduction, proliferation and mature. As well, several parameters affecting culture conditionswere optimized, and a procedure for the efficient induction and culture of somatic embryos ofTaxus cuspidata was established tentatively. The results are as follows:1. The formula of B₅+6-BA0.2mg·L^-1+NAA3.0mg·L^-1was proven to be the mostsuitable suspension culture medium for embryogenic callus induction by the young shootsTaxus cuspidata at22℃under darkness culture,90rpm,30g·L^-1sucrose in culture mediumwhich pH was5.8. The embryogenic callus induction period was about20～25d.2. The most suitable culture medium for embryogenic callus proliferation and maturationwas B₅+NAA2.0mg·L^-1+6-BA0.2mg·L^-1, at22℃under darkness, and shaking at90rpm,supplemented with30g·L^-1sucrose in the medium, and the pH was adjusted to5.8. After24dof culture, the callus was transferred to solid medium B₅+ABA3.0mg·L^-1at1500lx illum-ination for somatic embryo maturation until the end of the55thday.3. The results further indicated that illumination intensity could exert influence on thesomatic embryo induction and proliferation to a certain extent. In the late stage of somaticembryogenesis, it became yellowish and withered gradually under strong illumination, inaddition, imperfect embryos chloroplast occurred and led to immaturation of somatiocembryo under too weak illumination. According to the data, subjection to1500lx proved tobe useful for the development of somatic embryo in the late stage of embryogenesis.4. On one hand, the somatic embryo granules became browning in appearance underhigher rotation speed, and led more cells to death during sub-culture. On the other hand, morecells couldn’t grow and differentiate normally under low rotation speed. Hence, a rotation of 90rpm was identified to be a suitable parameter.5. At pH6.0and22℃, treated seeds were pre-cultured on the hormone-free media for10days, and then transferred to B₅+2.0mg·L^-1NAA+0.5mg·L^-12,4-D with400mg·L^-1glutamine, it could reach a maximum induction rate of78.36%.6.The results demonstrated that B₅+NAA1.0g·L^-1+2,4-D1.0mg·L^-1+6-BA0.1mg·L^-1with addition of400mg·L^-1glutamine was tested to be the suitable formular for the somatic embryos proliferation under darkness. The proliferation rate was85.35%in25-days, which was a proliferated period.7. The embryogenic callus was transferred to solid B₅with3.0mg·L^-1ABA for embryomaturation under1500lx illumination. After20-days, callus transfer to no-hormone mediumfor embryo maturation.